【作者】 韩为东；

【导师】 楼方定；

【作者基本信息】 军医进修学院 ， 血液内科， 2001， 博士

【摘要】 为寻找克隆与急性白血病（AL）复发密切相关的基因，本组曾采用限制性路 标基因组扫描技术对初诊与复发两种不同病程阶段的急性髓系白血病（AML）细 胞基因组 DNA进行了 CpG岛甲驯差异的比较；最终克隆到一段复发状态白血病 细胞中NotI酶切位点CpG高度甲基化的DNA片段（2948bp）。本研究从这一DNA 序列出发，通过GenBank nr及hESTs数据库电子杂交信息，采用 3’及 5’－cDNA 末端快速扩增技术（Rapid Amplification of cDNA End），克隆到一个与白血病 复发相关的人类基因全长编码 cDNA，本组将其命名为 LRP16,作为新基因于 1999 年12在国际GenBank首次登录注册（登录号：AF202922）。以 LRP16全长cDNA 序列为探针，惜助人类基因组测序信息将该基因精确定位于染色体11q12.1，在 该位置与 FLRT1、FLJ20113三个基因共同形成一个小的基因簇。序列分析表明 LRP16跨越8．8kb的基因组序列；包含11个外显子，启动子区域缺乏保守的TATA 盒，富含CpG岛。转录本长1225bp，GC含量67.5%, 同一读码框内有两个起始密 码子ATG，第二个的侧翼具有Kozak保守特征,故该基因可能编码产生眼端长 短不同的两条肽链，长型 325个氨基酸,短型 243个氨基酸。多组织 Northern Blot 杂交结果显示LRP16基因具有组织差异性表达,在卵巢及结肠粘膜中最高，前列 腺和小肠中居中,睾丸中较低，脾、胸腺及外周血白细胞表达最弱。在对其短型 编码链进行原核表达时,筛选到一种第四外显子缺失型剪接体。 为探讨LRP16在肿瘤发生、发展、以及在血细胞成熟过程中可能的作用，本 研究采用信息学和实验学结合的方法分析了 LRP16基因的 SAGE谱并检测了在造 血发育不同阶段的表达状况。首先以 NCBI提供的高通量CGAP/SAGEmap数据库为 实验对象，“Virtual Northern”程序为实验工具，分析比较了LRP16基因在多种 肿瘤组织、细胞系和相应正常组织中的表达谱，继而采用半定量RT－PCR方法检 测了该基因在多种正常血细胞及白血病细胞中的表达量。SAGE结果显示 LRP16在 3 一 人类多种肿瘤细胞中的表达量明显高于其正常对应组织。啪中未检测到 LRP 表达，正常骨髓xiZ G－CSF动员的 PBSC（PeriPheral Blood Ste。Cells）中的 表达量明显低于正常人外周血、原代白血病细胞及白血病细胞系（P的．001）。初 步表明 LRP与造血细胞发育相关，并可能随免疫成熟表达上调。同时表明 LRP 与急性白血病等多种肿瘤的发生、发展密切相关；在初诊与复发白血病中，LRP16 的表达差异值得进一步研究。 为阐明 LRPI6编码蛋白可能具有的生物学功能，本文采用计算机辅助对该基 因可能编码的长型、短型及剪接型肽链进行了序列相似性、直系同源、结构功能 域分析，发现各型蛋白具有一致的信息学预测结果，相似性检索发现P北．05的 序列几乎均是一些单链MA反转录病毒编码的与皿A相互作用的非结构蛋白。直 系同源检索提示 LRP16编码核酸结合蛋白；结构功能域扫描到三个相互包含的 。。tif：his。acro、DFU27、Alpp。在止匕基础上将其短型肽链编码区插A pEGFP－NI 载体；融合到绿色荧光蛋白的氨基端，转导HeLa细胞后瞬时表达，激光共聚焦 显溅（Confocal）观察结果显示绿色荧光密集分布于细胞核内。信息学与实验 结果结合进一步分析认为 LRP蛋白是一起源古远；进化速度缓慢的核园子，该 蛋白应在细胞多种生命行为中起重要作用；具体功能可能是一种磷酸酯酶。 总之，本研究克隆到一个与 AML复发密切相关的人类新基因，DNA的甲基化 极有可能是构成调控其表达的直接因素入RP在 AL及人类多数肿瘤中表达紊乱， 呈上调趋势。功能分析认为该基因应是一个在细胞多种生命行为活动中起重要作 用的核蛋白因子，并与造血细胞的发育高度相关，可能随淋巴细胞免疫成熟而上 调表达量。LRP在复发与初诊状态 AML白血病细胞中的表达差异值得进一步研 究。更多还原

【Abstract】 In order to find and clone relapse-related genes of acute leukemia (AL), we had ever compared the methylat ion difference of DNA CpG island between diagnostic and relapsed acute myeloid leukemia (AML) using restriction landmark genomic scanning technique. A 2948bp DNA fragment containing two Not I restriction enzyme sites had been cloned. Further research demonstrated that CpG within one Not I was significantly methylated in relapsed acute myeloid leukemia. In this research starting from this DNA fragment with the aid of electronic hybridization in nr and hEST (human Expression Sequence Tags) data bases, the complete coding cDNA sequence of a human gene which is associated with relapsed AML has been cloned employing 3’-and5’-RACE technique(Rapid Amplification of cDNA End ).After confirming it as a novel gene, our research group named it LRP16 and registered it in GenBank in 1999 December (Accession Number: AF202922) Subsequently, using its cDNA sequence and human genome sequencing information releasing by internet, LRP16 gene was exactly located on chromosome 11q12. 1. Within this subregion of chromosome 11q12. 1, one small gene cluster including LRP16, FLRT1, FLJ2O113 was observed. The gene LRP16 spans 8. 8kb on genomic DNA and contains 11 exons. The sequence of its promote region is CpG rich and lacks conserved TATA box. The full-length transcript of LRP16 is 1225bp and has high GC content (reaching to 67. 5%) Because two initiating codon ATG was observed within the same reading frame of LRP16 cDNA and the second context has nearly conserved Kozak feature, we postulated that LRP16 gene may translate two types of peptide chain, 5 the length type and the short . The length type has 325 amino acids and the short has 243aa, the only difference between these two is the Nerminal. The result of multi issue Northern blot hybridization displayed that LRP16 gene had different expression pattern in different tissues, highly in overy and colon mucosa, moderately in prostate and small intestine, lower in testis and lowest in spleen, thymus and peripheral blood. A fourthxoneleted splicing type was screened through the prokaryotic expression of LRP16 shortype coding chain using BL21 E.Coli bacterium, the expression amount of which is comparably lower in most tissues. To investigate the possible significance of LRP16 during tumor development and progression and during hematopoietic maturation, bioinformatic and experimental method were employed to detect the expression state of LRP16 in tumor development and different hematopoietic stages. Using CGAP/SAGEmap database provided by NCBI as experimental material, irtual Northern?program as tool, LRP16 expression pattern was compared among multiple tumor tissues, cell lines and corresponding normal tissues. Semiuantified RTCR was adopted to compare the expression amount among normal blood cells and leukemie cells. Results of SAGE showed that LRP16 gene was highly expressed in several human tumor cells than in their normal counterparts. LRP16 was not detected in cord blood cells. The expression amount of LRP16 gene in normal bone marrow and PBSC ( peripheral blood stem cells ) mobilized by GSF were significantly lower than in normal peripheral blood, priary leukemic cells and leukemic cell lines (P<0. 0001) suchasHL60, K562andU937. These results suggested that LRP16 is a gene associated with hematopoietic 6更多还原